Interferon regulatory factor 6 (IRF6) variants are associated with common isolated forms of cleft lip with or without clefting of the palate (CLP), as well as with dominantly inherited forms of CLP. Based on high sequence and structural homology with other IRFs, IRF6 has been predicted to act as a transcription factor, although studies in keratinocytes have found IRF6 primarily in the cytoplasm. Recent studies in the Cox laboratory have found that IRF6 binds, in a phosphorylation-enhanced manner, to NME2 (non-metastatic 2), which has been reported to interact with factors that activate the PAR (PARD3-PARD6) polarity complex. NME regulated PARD3 activation stimulates establishment of epithelial apical and basolateral domains (i.e. polarity), ultimately impacting epithelial cell adhesion, shape, and behavior. Thus, via protein-protein interactions, IRF6 is hypothesized to contribute to the regulation of epithelial polarity, which when disrupted, leads to CLP. Specific aims were developed to investigate the contribution of IRF6 to primary palate and tooth development. Using site-directed mutagenesis, reported VWS patient mutations will be introduced into yeast and mammalian expression constructs and subsequently compared with wild type IRF6 in their ability to interact with NME2 . Potential effects of the IRF6-NME2 interaction on epithelial polarity will be assessed by measuring activity of polarity effector proteins, assaying for nuclear:cytoplasmic ratios of polarity markers, and observing for epithelial morphological changes. To determine a relationship between IRF6-NME2 binding and primary palatal fusion, a novel in ovo procedure developed by the Cox laboratory will be used to specifically perturb IRF6/NME2 expression in embryonic chick orofacial epithelia, and effects on primary palatal development characterized. In human patients, CLP is often associated with tooth abnormalities, and in mice, IRF6 was detected in ameloblasts and has roles in tooth epithelial invagination. The tooth also presents another mechanism to investigate IRF6 contribution to epithelial polarity; ameloblasts are derived from the oral epithelium and exhibit observable polarity during the secretory stage. A murine model with IRF6 conditionally ablated in ameloblasts will be developed, and IRF6 epithelial polarity contribution will be evaluated based on changes in ameloblast polarity, enamel formation, and gene expression (laser capture microdissection followed by microarray analysis). Thus, these studies will also explore a possible relationship between CLP and tooth development. This proposed F30 project was developed to help provide a foundation for an aspiring dentist scientist and contribute to research centered on genetic and molecular causes of CLP. IRF6 has been targeted because of its prominent contribution to CLP incidence. Understanding regulatory mechanisms behind CLP will help identify susceptible populations, lower incidence, and develop treatment strategies. With strong institutional support from the University of Washington and excellent mentorship, we are confident that this F30 award will aid in the basic sciences and clinical training required to be a successful clinician scientist.